scholarly journals Posttranslational Modification of Bcl-2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1886-1896 ◽  
Author(s):  
Kristin Breitschopf ◽  
Judith Haendeler ◽  
Philipp Malchow ◽  
Andreas M. Zeiher ◽  
Stefanie Dimmeler
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Map Kinase ◽  
Specific Inhibitor ◽  
26S Proteasome ◽  
Apoptotic Pathway ◽  
Critical Determinant ◽  
Intact Cells ◽  
Antiapoptotic Protein ◽  
Tnf Α

ABSTRACT The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-α) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-α induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-α-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.

scholarly journals TNF-α-Induced YAP/TAZ Activity Mediates Leukocyte-Endothelial Adhesion by Regulating VCAM1 Expression in Endothelial Cells

2018 ◽  
Vol 19 (11) ◽  
pp. 3428 ◽  
Author(s):  
Hyun-Jung Choi ◽  
Na-Eun Kim ◽  
Byeong Kim ◽  
Miran Seo ◽  
Ji Heo
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Leukocyte Adhesion ◽  
Hippo Pathway ◽  
Specific Inhibitor ◽  
Vascular Diseases ◽  
Adhesive Properties ◽  
Sprouting Angiogenesis ◽  
Tnf Α

YAP/TAZ, a transcriptional co-activator of Hippo pathway, has emerged as a central player in vessel homeostasis such as sprouting angiogenesis and vascular barrier stabilization, during development. However, the role of YAP/TAZ in pathological angiogenesis remains unclear. Here, we demonstrated that YAP/TAZ is a critical mediator in leukocyte-endothelial adhesion induced by the vascular inflammatory cytokine TNF-α. YAP/TAZ was dephosphorylated, translocated from the cytosol to the nucleus, and activated by TNF-α in endothelial cells. A specific inhibitor of Rho GTPases suppressed the TNF-α-induced dephosphorylation of YAP. Knockdown of YAP/TAZ using siRNA significantly reduced the expression of the leukocyte adhesion molecule VCAM1 induced by TNF-α. The adhesion of monocytes to endothelial cells was also markedly reduced by YAP/TAZ silencing. However, knockdown of YAP/TAZ did not affect TNF-α-induced NF-κB signaling. Overall, these results suggest that YAP/TAZ plays critical roles in regulating TNF-α-induced endothelial cell adhesive properties without affecting the NF-κB pathway, and implicate YAP/TAZ as a potential therapeutic target for treating inflammatory vascular diseases.

scholarly journals Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding

1998 ◽  
Vol 18 (10) ◽  
pp. 5670-5677 ◽  
Author(s):  
Ossama Abu Hatoum ◽  
Shlomit Gross-Mesilaty ◽  
Kristin Breitschopf ◽  
Aviad Hoffman ◽  
Hedva Gonen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
26S Proteasome ◽  
Intact Cells ◽  
Free System ◽  
Ubiquitin System

ABSTRACT MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein.

Angiopoietin-2 promotes inflammatory lymphangiogenesis and its effect can be blocked by the specific inhibitor L1-10

2012 ◽  
Vol 302 (1) ◽  
pp. H215-H223 ◽  
Author(s):  
Zhi-Xin Yan ◽  
Zhao-Hua Jiang ◽  
Ning-Fei Liu
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Transplant Rejection ◽  
Lymphatic Vessels ◽  
Specific Inhibitor ◽  
Biological Behavior ◽  
Tnf Α ◽  
Inflammatory Stimuli ◽  
Angiopoietin 2

Angiopoietin (Ang)-2, a ligand of the receptor tyrosine kinase Tie2, is known to be involved in the regulation of embryonic lymphangiogenesis. However, the role of Ang-2 in postnatal pathological lymphangiogenesis, such as inflammation, is largely unknown. We used a combination of imaging, molecular, and cellular approaches to investigate whether Ang-2 is involved in inflammatory lymphangiogenesis. We observed strong and continuous expression of Ang-2 on newly generated lymphatic vessels for 2 wk in sutured corneas of BALB/c mice. This expression was concurrent with an increased number of lymphatic vessels. TNF-α expression also increased, with peak TNF-α expression occurring before peak Ang-2 expression was reached. In vitro experiments showed that TNF-α stimulates Ang-2 and Tie2 and ICAM-1 expression on human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs). Ang-2 alone did not affect the biological behavior of LECs, whereas Ang-2 combined with TNF-α significantly promoted the proliferation of LECs but not BECs. In mouse models, blockade of Ang-2 with L1-10, an Ang-2-specific inhibitor, significantly inhibited lymphangiogenesis but promoted angiogenesis. These results clearly indicate that Ang-2 acts as a crucial regulator of inflammatory lymphangiogenesis by sensitizing the lymphatic vasculature to inflammatory stimuli, thereby directly promoting lymphangiogenesis. The involvement of Ang-2 in inflammatory lymphangiogenesis provides a strong rationale for the exploitation of anti-Ang-2 treatment in the prevention and treatment of tumor metastasis and transplant rejection.

scholarly journals Disturbed flow: p53 SUMOylation in the turnover of endothelial cells

2011 ◽  
Vol 193 (5) ◽  
pp. 805-807 ◽  
Author(s):  
Wakako Takabe ◽  
Noah Alberts-Grill ◽  
Hanjoong Jo
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Protein Inhibitor ◽  
Disturbed Flow ◽  
Antiapoptotic Protein ◽  
Kinase C

Disturbed blood flow induces apoptosis of vascular endothelial cells, which causes atherosclerosis. In this issue, Heo et al. (2011. J. Cell Biol. doi:10.1083/jcb.201010051) sheds light on p53’s role in this phenomenon. Disturbed flow induces peroxynitrite production, which activates protein kinase C ζ and it’s binding to the E3 SUMO (small ubiquitin-like modifier) ligase PIASy (protein inhibitor of activated STATy). This leads to p53 SUMOylation and its export to the cytosol, where it binds to the antiapoptotic protein Bcl-2 to induce apoptosis.

scholarly journals Dephosphorylation Targets Bcl-2 for Ubiquitin-dependent Degradation: A Link between the Apoptosome and the Proteasome Pathway

1999 ◽  
Vol 189 (11) ◽  
pp. 1815-1822 ◽  
Author(s):  
Stefanie Dimmeler ◽  
Kristin Breitschopf ◽  
Judith Haendeler ◽  
Andreas M. Zeiher
Keyword(s):  
Endothelial Cells ◽  
Map Kinase ◽  
Inflammatory Diseases ◽  
Apoptotic Cell ◽  
Proteasome Inhibitors ◽  
Inhibitory Function ◽  
Tnf Α ◽  
Proteasome Pathway

Injury of the endothelial cells by the induction of apoptotic cell death may play an important role in the pathophysiology of atherosclerosis and the progression of inflammatory diseases. Here, we demonstrate an essential role for the ubiquitin-dependent proteasome complex in stimulus-induced degradation of the antiapoptotic protein Bcl-2. Bcl-2 is specifically degraded after stimulation of human endothelial cells with tumor necrosis factor (TNF)-α in a process that is inhibited by specific proteasome inhibitors. In addition, the mutation of the potential ubiquitin-acceptor amino acids of Bcl-2 provides protection against TNF-α– and staurosporine-induced degradation in vitro and in vivo. Moreover, mimicking phosphorylation of the putative mitogen-activated protein (MAP) kinase sites of the Bcl-2 protein (Thr 56, Thr 74, and Ser 87) abolishes its degradation, suggesting a link between the MAP kinase pathway to the proteasome pathway. Finally, inhibition of Bcl-2 degradation either by suppressing ubiquitin-dependent proteasomal degradation or by mimicking continuous phosphorylation of the putative MAP kinase sites in the Bcl-2 protein confers resistance against induction of apoptosis. Thus, the degradation of Bcl-2 may unleash the inhibitory function of Bcl-2 over the apoptosome and may thereby amplify the activation of the caspase cascade.

Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells

2021 ◽  
Vol 53 (5) ◽  
pp. 567-574
Author(s):  
Weiwei Zeng ◽  
Zhiyuan Sun ◽  
Tengxiang Ma ◽  
Xiaobin Song ◽  
Shuai Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Umbilical Vein ◽  
Specific Inhibitor ◽  
Critical Event ◽  
Necrosis Factor Alpha ◽  
Interacting Protein ◽  
Multiple Substrates ◽  
Leucocyte Adhesion ◽  
Tnf Α

Abstract Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury. This process is primarily regulated by the expression of cell adhesion molecules (CAMs) in endothelial cells. It has been well documented that tumor necrosis factor alpha (TNF-α) is a key regulator of CAM expression within this process, but its regulatory mechanism remains controversial. To investigate the scenario within this process, we assessed the role of zipper-interacting protein kinase (ZIPK), a serine/threonine kinase with multiple substrates, in CAM expression. We used TNF-α as inflammatory stimulator and found that ZIPK was integrated into the signaling regulation of TNF-α-mediated CAM expression. In human umbilical vein endothelial cells (HUVECs), TNF-α exposure led to significantly increased expression of both intercellular CAM-1 (ICAM-1) and vascular CAM-1 (VCAM-1), along with an increase in the adhesion of THP-1 monocytes to HUVECs. Simultaneously, ZIPK gene was also up-regulated at the transcription level. These effects were clearly inhibited by the ZIPK-specific inhibitor Tc-DAPK6 or small interfering RNA (siRNA) capable of specifically inhibiting ZIPK expression. We thus suggest that both ZIPK activation and ZIPK gene expression are necessary for TNF-α-mediated CAM expression and leucocyte adhesion. Interestingly, ZIPK inhibition also significantly suppressed TNF-α-induced nuclear factor kappa B (NF-κB) activation, indicating that TNF-α-mediated ZIPK expression functions upstream of NF-κB and CAM expression. We thus propose a TNF-α/ZIPK/NF-κB signaling axis for CAM expression that is necessary for leucocyte adhesion to endothelial cells. Our data in this study revealed a potential molecular target for exploring anti-inflammation drugs.

Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression

2006 ◽  
Vol 290 (5) ◽  
pp. H1862-H1870 ◽  
Author(s):  
Xi-Lin Chen ◽  
Geraldine Dodd ◽  
Suzanne Thomas ◽  
Xiaolan Zhang ◽  
Martin A. Wasserman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Map Kinase ◽  
P38 Map Kinase ◽  
Control Element ◽  
Related Factor ◽  
Dependent Manner ◽  
Inflammatory Gene Expression ◽  
Inflammatory Gene ◽  
Tnf Α

The antioxidant response element (ARE) is a transcriptional control element that mediates expression of a set of antioxidant proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that activates ARE-containing genes. In endothelial cells, the ARE-mediated genes are upregulated by atheroprotective laminar flow through a Nrf2-dependent mechanism. We tested the hypothesis that activation of ARE-regulated genes via adenovirus-mediated expression of Nrf2 may suppress redox-sensitive inflammatory gene expression. Expression of Nrf2 in human aortic endothelial cells (HAECs) resulted in a marked increase in ARE-driven transcriptional activity and protected HAECs from H2O2-mediated cytotoxicity. Nrf2 suppressed TNF-α-induced monocyte chemoattractant protein (MCP)-1 and VCAM-1 mRNA and protein expression in a dose-dependent manner and inhibited TNF-α-induced monocytic U937 cell adhesion to HAECs. Nrf2 also inhibited IL-1β-induced MCP-1 gene expression in human mesangial cells. Expression of Nrf2 inhibited TNF-α-induced activation of p38 MAP kinase. Furthermore, expression of a constitutively active form of MKK6 (an upstream kinase for p38 MAP kinase) partially reversed Nrf2-mediated inhibition of VCAM-1 expression, suggesting that p38 MAP kinase, at least in part, mediates Nrf2's anti-inflammatory action. In contrast, Nrf2 did not inhibit TNF-α-induced NF-κB activation. These data identify the Nrf2/ARE pathway as an endogenous atheroprotective system for antioxidant protection and suppression of redox-sensitive inflammatory genes, suggesting that targeting the Nrf2/ARE pathway may represent a novel therapeutic approach for the treatment of inflammatory diseases such as atherosclerosis.

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